作　　者： (沈天成）; (白亚军）; (郑晓辉）; (蔡宇杰）;

机构地区： 江南大学生物工程学院工业生物技术教育部重点实验室,江苏无锡214122

出　　处： 《生物技术》 2017年第4期383-389,共7页

摘　　要： [目的]对奇异变形杆菌JN458 D-乳酸氧化酶进行酶学性质研究。[方法]克隆D-乳酸氧化酶基因(lod D),构建重组质粒在大肠中表达,生物信息学初步分析D-乳酸氧化酶氨基酸序列,镍柱纯化该酶并研究其性质。[结果]生物信息学分析该酶C-末端含膜结合结构域,所以是膜蛋白,属于以醌作为电子受体的D-乳酸氧化酶。最适温度60℃,50℃下保持稳定1 h,中高温适应性较好。最适pH 7,pH 7~9下保持稳定1 h。以D-乳酸为底物时Km值为0.61 mmol/L,对D-乳酸有很高的亲和力。Fe^(2+)、Mn^(2+)和Mg^(2+)在1~6 mmol/L的浓度内能提高酶活,其中Fe^(2+)效果最好。[结论]lod D在大肠中实现了高效表达,使新型D-乳酸氧化酶的应用有了理论依据。 [Objectives]D-lactate oxidase derived from Proteus mirabilis JN458 was chacacterized. [Methods]The D-lactate oxidase gene（ lod D） was cloned and the recombinant plasmid was constructed to express in E. coli at first,then the amino acid sequence of the enzyme was initially analyzed bioinformatically,finally the enzyme was purified by nickel column and chacacterized. [Results] The enzyme was predicted to have a membrane-bound domain at its C-terminal by bioinformatics analysis,so it was a membrane protein and took the quinone as an electron acceptor. The optimal temperature of the enzyme was determined to be 60℃ and the enzyme was stable at 50℃ when stored for 1 hour at pH 7,so it had a good medium and high temperature adaptability. The optimal pH of the enzyme was determined to be 7 and it was stable across a pH range of 7-9when stored for 1 h at 30 ℃. Kmvalue for D-lactate was 0. 61 mmol/L,which indicated the enzyme had a high affinity for D-lactate. Enzyme activity was enhanced by Fe^（2＋）,Mn^（2＋）and Mg^（2＋）in the range of 1-6 mmol/L,while Fe^（2＋）had the best effect.[Conclusion]The high expression of lod D was achieved in E. coli for the first time,which provided a theoretical basis for the application of the noval D-lactate oxidase.

关 键 词： 奇异变形杆菌 乳酸氧化酶 酶学性质 生物信息学分析 高效表达